709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

SummaryManganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health.Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Constructed subsurface-flow wetlands are becoming more common for on-site treatment of domestic wastewater to provide secondary quality effluent. Macrophytes are generally added to wetlands to increase treatment efficiency but their role in reducing N content is controversial. Our investigation utilized two subsurface-flow wetlands to determine the efficiency of different macrophytes in uptake of 15N labeled ammonium sulfate. Macrophytes in Wetland 1 recovered 35% of the added N in their shoots but only 5% of the added N was recovered in the shoots and roots in Wetland 2. A major difference for the two wetlands was N and hydraulic loadings. Wetland 1 received 7.5 Kg N ha(-1)d(-1) and Wetland 2 received 16.9 Kg N ha(-1)d(-1). Retention time for Wetland 1 based on pore volumes was 2.9 d and for Wetland 2 it was 1.2 d. The retardation factor for NH4+ was approximately 2.5 for both wetlands and the breakthrough curves indicated lack of plug flow. The importance of macrophytes in taking up NH4 appeared to dependent on N and hydraulic loadings.